Compositions and methods for nucleic acid extraction

ABSTRACT

Compositions and techniques for the extraction, enrichment and isolation of nucleic acids from yeast in a whole blood sample using amine monomers are disclosed herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 16, 2014, isnamed 01886-2008US03_SL.txt and is 1,727 bytes in size.

BACKGROUND

Prior art methods for nucleic acid extraction from cellular sourcematerials and, particularly, paraffin embedded tissue samples (e.g.,formalin-fixed paraffin-embedded samples: FFPE) involves complicated,multi-step processes.

The extraction of nucleic acids from Mycobacteria in sputum, forexample, is a challenge because sputum is very viscous and not easilyprocessed for nucleic acid extraction. Sputum samples are typicalsolubilized using N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH)treatment (Coulter and Charache, Sputum digestion/decontamination forMycobacteriology culture—Guidelines, SMILE, John Hopkins University,2008) and the mycobacteria are pelleted by centrifugation. NALC-NaOHtreatment does not kill the Mycobacteria and further treatment by heatand/or chemicals is done to inactivate the samples. Nucleic acids can beextracted from the cell pellet using several techniques to lyse cells.Sonication (Colin, et al., Method and apparatus for ultrasonic lysis ofbiological cells, U.S. Pat. No. 6,686,195, 2004), bead beating(Melendes, et al., Cell disrupting apparatus, U.S. Pat. No. 5,464,773,1995), enzymes (Salazar and Asenjo, Enzymatic lysis of microbial cells,Biotechnol Lett (2007) 29:985-994), mixing (vortexing), mechanicalshearing and chaotropic solutions (Das, et al., Method for detectingpathologenic mycobacteria in clinical specimens, U.S. Pat. No.7,638,309, 2009) are some of the methods used to break open the pelletedcells for nucleic acid extraction. These steps are in addition to theactual extraction procedures and add complexity to and time to theentire process.

The extraction of nucleic acid from yeast is also one of the morechallenging techniques in nucleic acid (e.g., DNA) sample preparation.Yeast are fungi and have cell walls that are difficult to lyse (Lipkeand Ovalle, Cell wall architecture in yeast: new structure and newchallenges, J Bacteriol 1998, 180(15):3735). Lysis buffers usingchaotropic salts and detergents or alkali lysis protocols of the priorart are not very effective in lysing yeast cells directly but are usedwith additional steps. These additional steps can be divided into twomain groups: physical methods and enzymatic methods. The physicalmethods can include sonication of cells (U.S. Pat. No. 6,686,195) withor without the presence of grinding particles, high powered agitationwith grinding particles (U.S. Pat. No. 5,464,773) (bead beating, ballmills) or the use of high pressure mechanical shearing (e.g., Frenchpressure cell press, as is known in the art). Enzymatic methods rely onparticular enzymes such as zymolase (Salazar and Asenjo, ibid; U.S. Pat.No. 5,688,644) to weaken the cell walls such that the cells can be lysedby more conventional techniques.

The extraction, enrichment and isolation of nucleic acids from FFPEmaterial is a very complicated process that requires thedeparaffinization of the tissue with organic solvents, the digestion ofthe tissue with protease and then the extraction of the nucleic acidsfrom the tissue. These prior art processes use multiple solutions andmultiple steps. The organic solvents used are not usually miscible withaqueous solutions.

Thus, what is needed are compositions and methods that permit theefficient extraction, enrichment, isolation and purification of nucleicacids from cellular source materials, particularly Mycobacteria, yeastand FFPE samples.

SUMMARY OF THE INVENTION

The present invention solves the prior art problem of nucleic acidextraction by providing a one-step method for the extraction of nucleicacids from cellular source material including bacteria, yeast andformalin-fixed paraffin-embedded (FFPE) tissue. In one embodiment, thepresent invention comprises an aqueous extraction solution capable oflysing cells and purifying nucleic acid in one step.

The present invention is directed towards a nucleic acid extractionmethod that allows the direct extraction of nucleic acids from samplesincluding FFPE tissue. This method utilizes a combination of polar andnon-polar organic solvents as well as chaotropes and detergents tosolubilize the paraffin, break down the tissue and release the nucleicacids. The nucleic acids then can be, for example, captured on silicacontaining particles in a single solution. Capture particles, if used,may be magnetic. There are no separate de-paraffinization steps orprotease digestions in this process. The organic solvents are completelymiscible and there is no phase separation in this process. Theextraction method uses amine monomers such as2,2′-(ethylenedioxy)bis(ethylamine) in combination with an aqueoussolution containing a chaotrope such as urea or guanidine thiocyanateand detergent. The extraction method may also contain, optionally, otherorganic solvents such as dimethyl sulfoxide (DMSO), various alcohols,and limonene. The process is extremely simple. The sample, (e.g., a FFPEtissue sample) is mixed with the extraction buffer containing the aminemonomer (e.g., one or more of 2,2′-eththylenedioxy)bis(ethylamine)(EDBE), 1,3-diaminopropane (DAP), 2-amino-1-butanol (AB),2-(2-aminoethoxy)ethanol (AEE), 2-amino-6-methylheptane (AMH),2-amino-2-methyl-1-propanol (AMP), amino-2-propanol (A2P),1,5-diamino-2-methylpentane (DMP) and 3-amino-1-propanol (3A1P)). Themixture may optionally be warmed or heated to aid in the release of thenucleic acids. The nucleic acids may be captured on microparticles (orother suitable solid substrate known to one of ordinary skill in theart). For example, silica coated magnetic particles may be added to themixture and the nucleic acids captured on the particles. Other methodsof capturing the nucleic acids that are known to one of ordinary skillin the art are also suitable for use in the present invention. Noadditional solutions are needed and there are no deparaffinization stepsor protease digestions. The particles are washed (or otherwiseprocessed) to remove any impurities and the nucleic acids are releasedfrom the silica particles with water or a dilute buffer solution.

With regard to the enrichment, extraction, isolation and purification ofnucleic acids from other cellular source samples such as, but notlimited to, Mycobacteria and yeast, the methods and compositionssummarized above are also suitable.

An advantage of the present invention is that, unlike prior art methods,the extraction of the nucleic acid from the subject sample does notrequire the use of enzymes for the lysis of the cellular material.

The present invention contemplates a method of extracting nucleic acidfrom cellular source material, said method comprising: providing i)cellular source material and ii) an aqueous extraction solutioncomprising one or more amine monomers; contacting said cellular sourcematerial with said extraction solution resulting in lysis of thecellular material and extraction of the nucleic acids. The presentinvention also contemplates that amine monomer is a primary aminemonomer. The present invention further contemplates that the aminemonomer is one or more of 2,2′-eththylenedioxy)bis(ethylamine) (EDBE),1,3-diaminopropane (DAP), 2-amino-1-butanol (AB),2-(2-aminoethoxy)ethanol (AEE), 2-amino-6-methylheptane (AMH),2-amino-2-methyl-1-propanol (AMP), amino-2-propanol (A2P),1,5-diamino-2-methylpentane (DMP) and 3-amino-1-propanol (3A1P). It isfurther contemplated that the aqueous extraction solution may comprise achaotrope and that the chaotrope is selected from one or more of thegroup consisting of urea, guanidine thiocyanate (GITC), ethanol andbutanol. It is further contemplated that the aqueous extraction solutionmay comprise one or more of a detergent and an alcohol and the thatdetergent may be selected from one or more of the group consisting ofTween™, polysorbates, deoxycholate, sodium deoxycholate and sodiumdodecyl sulfate (SDS), NP-40 and Triton™ X-100. Further, the finalconcentration of said detergent may about 1% to 15%, about 8% to about15% or about 10%. If it further contemplated that the alcohol isselected from one or more of the group consisting of ethanol and butanoland that the final concentration of the alcohol is about 10% to about40% or about 20 to about 35%.

It is contemplated that the concentration of amine monomer in saidaqueous extraction solution is about 15% to about 50% or about 20% toabout 45%. It is further contemplated that the concentration of thechaotrope in said aqueous extraction solution is about 4 M to about 5 M.

The present invention contemplates that the source material may beselected from living cellular source material and fixed cellular sourcematerial. Further, it is contemplated that the living cellular sourcematerial may comprise a suspension of cells and, in some embodiments,the suspension of single comprise bacteria. The bacteria may compriseMycobacteria. In other embodiments, the suspension of cells may compriseyeast.

It is further contemplated that the aqueous extraction solution of thepresent invention may be enzyme-free and, further, may be protease-free.

It is further contemplated that the aqueous extraction solutionpreferably has a pH of about 10 to about 13 and more preferably a pH ofabout 12 to about 13.

It is further contemplated that fixed cellular source material maycomprise formalin-fixed paraffin embedded (FFPE) material.

The present invention contemplates an aqueous extraction solutionsuitable for the extraction of nucleic acids from cellular sourcematerial, said composition comprising one or more amine monomers; one ormore chaotropic reagents, one or more detergents and one or more organicsolvents. It is further contemplated that the amine monomer is a primaryamine monomer and that the amino monomer may be selected from one ormore of 2,2′-eththylenedioxy)bis(ethylamine) (EDBE), 1,3-diaminopropane(DAP), 2-amino-1-butanol (AB), 2-(2-aminoethoxy)ethanol (AEE),2-amino-6-methylheptane (AMH), 2-amino-2-methyl-1-propanol (AMP),amino-2-propanol (A2P), 1,5-diamino-2-methylpentane (DMP) and3-amino-1-propanol (3A1P). It is further contemplated that concentrationof amine monomer in the aqueous extraction solution is about 15% toabout 50% or about 20% to about 45%. Further, it is contemplated thechaotrope may be selected from one or more of the group consisting ofurea, guanidine thiocyanate (GITC), ethanol and butanol. It is furthercontemplated that the concentration of the chaotrope in the aqueousextraction solution is about 4 M to about 5 M. It is furthercontemplated that detergent may be selected from one or more of thegroup consisting of Tween™, polysorbates, deoxycholate, sodiumdeoxycholate and sodium dodecyl sulfate (SDS), NP-40 and Triton™ X-100and the final concentration may be about 1% to 15%, about 8% to about15% or about 10%. It is further contemplated that the alcohol may beselected from one or more of the group consisting of ethanol and butanoland that the final concentration of the alcohol may be about 10% toabout 40% or about 20 to about 35%.

The present invention contemplates a method of extracting nucleic acidfrom cellular source material, said method comprising: providing i)cellular source material and ii) an aqueous extraction solutioncomprising ammonium hydroxide; contacting said cellular source materialwith said extraction solution resulting in lysis of the cellularmaterial and extraction of the nucleic acids. The present inventionfurther contemplates said aqueous extraction solution further comprisesone or more chaotropes. The present invention further contemplates thatchaotrope is selected from one or more of the group consisting of urea,guanidine thiocyanate (GITC), ethanol and butanol. The present inventionfurther contemplates that the aqueous extraction solution furthercomprises one or more of a detergent and an alcohol. The presentinvention further contemplates that the detergent is selected from oneor more of the group consisting of Tween™, polysorbates, deoxycholate,sodium deoxycholate and sodium dodecyl sulfate (SDS), NP-40 and Triton™X-100 and that the concentration of the detergent is about 1% to 15%,about 8% to about 15% or about 10%. The present invention furthercontemplates that the alcohol is selected from one or more of the groupconsisting of ethanol and butanol and that the concentration of thealcohol is about 10% to about 40% or about 20 to about 35%. The presentinvention further contemplates that the concentration of the chaotropein the aqueous extraction solution is about 4 M to about 5 M. Thepresent invention further contemplates that the cellular source materialis selected from living cellular source material and fixed cellularsource material. The present invention further contemplates that theliving cellular source material comprises a suspension of single cells.The present invention further contemplates that the suspension of cellscomprise bacteria. The present invention further contemplates that thebacteria are Mycobacteria. The present invention further contemplatesthat the suspension of cells comprise yeast. The present inventionfurther contemplates that the aqueous extraction solution isenzyme-free. The present invention further contemplates that the aqueousextraction solution is protease-free. The present invention furthercontemplates that the fixed cellular source material comprisesformalin-fixed paraffin embedded (FFPE) material.

The present invention contemplates a method of extracting nucleic acidfrom cellular source material, said method comprising: providing i)cellular source material and ii) an aqueous extraction solutioncomprising one or more of urea and guanidine thiocyanate (GITC);contacting said cellular source material with said extraction solutionresulting in lysis of the cellular material and extraction of thenucleic acids. The present invention further contemplates that theaqueous extraction solution further comprises one or more of a detergentand an alcohol. The present invention further contemplates that thedetergent is selected from one or more of the group consisting ofTween™, polysorbates, deoxycholate, sodium deoxycholate and sodiumdodecyl sulfate (SDS), NP-40 and Triton™ X-100 and that theconcentration of said detergent is about 1% to 15%, about 8% to about15% or about 10%. The present invention further contemplates that thealcohol is selected from one or more of the group consisting of ethanoland butanol and that the concentration of said alcohol is about 10% toabout 40% or about 20 to about 35%. The present invention furthercontemplates that the total concentration of the one or more of urea andguanidine thiocyanate (GITC) in said aqueous extraction solution isabout 4 M to about 5 M. The present invention further contemplates thatthe cellular source material is selected from living cellular sourcematerial and fixed cellular source material. The present inventionfurther contemplates that the living cellular source material comprisesa suspension of single cells. The present invention further contemplatesthat the suspension of cells comprise bacteria. The present inventionfurther contemplates that the bacteria are Mycobacteria. The presentinvention further contemplates that the suspension of cells compriseyeast. The present invention further contemplates that the aqueousextraction solution is enzyme-free. The present invention furthercontemplates that the aqueous extraction solution is protease-free. Thepresent invention further contemplates that the fixed cellular sourcematerial comprises formalin-fixed paraffin embedded (FFPE) material. Theaqueous extraction solution of the present invention this embodiment mayalso comprise an amine monomer at a concentration of 15% to about 50% orabout 20% to about 45%.

The present invention contemplates a method of inactivating and killingMycobacterium, said method comprising: providing i) cellular sourcematerial comprising Mycobacterium and ii) an aqueous extraction solutioncomprising one or more amine monomers; contacting said cellular sourcematerial with said extraction solution resulting in lysis of thecellular material and the extraction of the nucleic acids. The presentinvention further contemplates that the amine monomer is a primary aminemonomer. The present invention further contemplates that the aminemonomer is 2,2′-(ethylenedioxy)bis(ethylamine) (EDBE). The presentinvention further contemplates that the amine monomer is1,3-diaminopropane. The present invention further contemplates that theamine monomer is 3-amino-1-propanol. The present invention furthercontemplates that the amine monomer is 2-amino-1-butanol (AB),2-(2-aminoethoxy)ethanol (AEE), 2-amino-6-methylheptane (AMH),2-amino-2-methyl-1-propanol (AMP), amino-2-propanol (A2P) or1,5-diamino-2-methylpentane (DMP). The present invention furthercontemplates that the aqueous extraction solution further comprises achaotrope. The present invention further contemplates that the chaotropeis selected from one or more of the group consisting of urea, guanidinethiocyanate (GITC), ethanol and butanol. The present invention furthercontemplates that the aqueous extraction solution further comprises oneor more of a detergent and an alcohol. The present invention furthercontemplates that the detergent is selected from one or more of thegroup consisting of Tween™, polysorbates, deoxycholate, sodiumdeoxycholate and sodium dodecyl sulfate (SDS), NP-40 and Triton™ X-100.The present invention further contemplates that the concentration ofsaid detergent is about 1% to 15%, about 8% to about 15% or about 10%.The present invention further contemplates that the alcohol is selectedfrom one or more of the group consisting of ethanol and butanol. Thepresent invention further contemplates that the concentration of saidalcohol is about 10% to about 40% or about 20 to about 35%. The presentinvention further contemplates that the concentration of amine monomerin said aqueous extraction solution is about 15% to about 50% or about20% to about 45%. The present invention further contemplates that theconcentration of the chaotrope in said aqueous extraction solution isabout 4 M to about 5 M. The present invention further contemplates thatthe source material comprising Mycobacterium comprises a suspension ofsingle cells. The present invention further contemplates that theaqueous extraction solution is enzyme-free. The present inventionfurther contemplates that the aqueous extraction solution isprotease-free. The present invention further contemplates that themethod further extracts nucleic acid from said Mycobacterium.

The present invention also contemplates that the source material maycomprise samples previously assayed by fluorescent in situ hybridization(FISH). FISH is known to those of ordinary skill in the art. FISHanalysis can be used to, for example, prescreen for targets or as acompanion assay. Samples positive for targets can then be quantified byextraction of the nucleic acid with the compositions and procedures ofthe present invention followed by, for example, PCR.

In one embodiment, the present invention contemplates a method ofextracting nucleic acid from cellular source material, said methodcomprising: providing i) cellular source material and ii) an aqueousextraction solution comprising one or more amine monomers; contactingsaid cellular source material with said extraction solution resulting inlysis of the cellular material and extraction of the nucleic acids. Thepresent invention further contemplates, that the amine monomer is aprimary amine monomer. The present invention further contemplates, thatthe amine monomer is 2,2′-(ethylenedioxy)bis(ethylamine) (EDBE). Thepresent invention further contemplates, that the amine monomer isselected from one or more of 1,3-diaminopropane and 3-amino-1-propanol.The present invention further contemplates, that the aqueous extractionsolution further comprises a chaotrope. The present invention furthercontemplates, that the chaotrope is selected from one or more of thegroup consisting of urea, guanidine thiocyanate (GITC), ethanol andbutanol. The present invention further contemplates, that the aqueousextraction solution further comprises one or more of a detergent and analcohol. The present invention further contemplates, that the detergentis selected from one or more of the group consisting of Tween™,polysorbates, deoxycholate, sodium deoxycholate and sodium dodecylsulfate (SDS), NP-40 and Triton™ X-100. The present invention furthercontemplates, that the detergent is about 8% v/v to about 15% v/v. Thepresent invention further contemplates, that the alcohol is selectedfrom one or more of the group consisting of ethanol and butanol. Thepresent invention further contemplates, that the concentration of saidalcohol is about 15% v/v to about 25% v/v. The present invention furthercontemplates, that the concentration of amine monomer in said aqueousextraction solution is about 30% v/v to about 50% v/v. The presentinvention further contemplates, that the concentration of the chaotropein said aqueous extraction solution is about 4 M to about 5 M. Thepresent invention further contemplates, that the cellular sourcematerial is selected from living cellular source material and fixedcellular source material. The present invention further contemplates,that the living cellular source material comprises a suspension ofsingle cells. The present invention further contemplates, that thesuspension of cells comprises bacteria. The present invention furthercontemplates, that the bacteria are Mycobacteria. The present inventionfurther contemplates, that the suspension of cells comprises yeast. Thepresent invention further contemplates, that the aqueous extractionsolution is enzyme-free. The present invention further contemplates,that the aqueous extraction solution is protease-free. The presentinvention further contemplates, that the fixed cellular source materialcomprises formalin-fixed paraffin embedded (FFPE) material.

The present invention contemplates a composition comprising an aqueousextraction solution suitable for the extraction of nucleic acids fromcellular source material, said composition comprising one or more aminemonomers; one or more chaotropic reagents, one or more detergents andone or more organic solvents. The present invention furthercontemplates, that the amine monomer is a primary amine monomer. Thepresent invention further contemplates, that the amine monomer is2,2′-(ethylenedioxy)bis(ethylamine) (EDBE). The present inventionfurther contemplates, that the amine monomer is selected from one ormore of 1,3-diaminopropane and 3-amino-1-propanol. The present inventionfurther contemplates, that the aqueous extraction solution furthercomprises a chaotrope. The present invention further contemplates, thatthe chaotrope is selected from one or more of the group consisting ofurea, guanidine thiocyanate (GITC), ethanol and butanol. The presentinvention further contemplates, that the aqueous extraction solutionfurther comprises one or more of a detergent and an alcohol. The presentinvention further contemplates, that the detergent is selected from oneor more of the group consisting of Tween™, polysorbates, deoxycholate,sodium deoxycholate and sodium dodecyl sulfate (SDS), NP-40 and Triton™X-100. The present invention further contemplates, that the detergent isabout 8% v/v to about 15% v/v. The present invention furthercontemplates, that the alcohol is selected from one or more of the groupconsisting of ethanol and butanol. The present invention furthercontemplates, that the concentration of said alcohol is about 15% v/v toabout 25% v/v. The present invention further contemplates, that theconcentration of amine monomer in said aqueous extraction solution isabout 30% v/v to about 50% v/v. The present invention furthercontemplates, that the concentration of the chaotrope in said aqueousextraction solution is about 4 M to about 5 M.

The present invention contemplates a method of extracting nucleic acidfrom cellular source material, said method comprising: providing i)cellular source material and ii) an aqueous extraction solutioncomprising ammonium hydroxide; contacting said cellular source materialwith said extraction solution resulting in lysis of the cellularmaterial and extraction of the nucleic acids. The present inventionfurther contemplates, that the aqueous extraction solution furthercomprises a chaotrope. The present invention further contemplates, thatthe chaotrope is selected from one or more of the group consisting ofurea, guanidine thiocyanate (GITC), ethanol and butanol. The presentinvention further contemplates, that the aqueous extraction solutionfurther comprises one or more of a detergent and an alcohol. The presentinvention further contemplates, that the detergent is selected from oneor more of the group consisting of Tween™, polysorbates, deoxycholate,sodium deoxycholate and sodium dodecyl sulfate (SDS), NP-40 and Triton™X-100. The present invention further contemplates, that the detergent isabout 8% v/v to about 15% v/v. The present invention furthercontemplates, that the alcohol is selected from one or more of the groupconsisting of ethanol and butanol. The present invention furthercontemplates, that the concentration of said alcohol is about 15% v/v toabout 25% v/v. The present invention further contemplates, that theconcentration of the chaotrope in said aqueous extraction solution isabout 4 M to about 5 M. The present invention further contemplates, thatthe cellular source material is selected from living cellular sourcematerial and fixed cellular source material. The present inventionfurther contemplates, that the living cellular source material comprisesa suspension of single cells. The present invention furthercontemplates, that the suspension of cells comprises bacteria. Thepresent invention further contemplates, that the bacteria areMycobacteria. The present invention further contemplates, that thesuspension of cells comprises yeast. The present invention furthercontemplates, that the aqueous extraction solution is enzyme-free. Thepresent invention further contemplates, that the aqueous extractionsolution is protease-free. The present invention further contemplates,that the fixed cellular source material comprises formalin-fixedparaffin embedded (FFPE) material.

The present invention contemplates a method of extracting nucleic acidfrom cellular source material, said method comprising: providing i)cellular source material and ii) an aqueous extraction solutioncomprising one or more of urea and guanidine thiocyanate (GITC);contacting said cellular source material with said extraction solutionresulting in lysis of the cellular material and extraction of thenucleic acids. The present invention further contemplates, that theaqueous extraction solution further comprises one or more of a detergentand an alcohol. The present invention further contemplates, that thedetergent is selected from one or more of the group consisting ofTween™, polysorbates, deoxycholate, sodium deoxycholate and sodiumdodecyl sulfate (SDS), NP-40 and Triton™ X-100. The present inventionfurther contemplates, that the detergent is about 8% v/v to about 15%v/v. The present invention further contemplates, that the alcohol isselected from one or more of the group consisting of ethanol andbutanol. The present invention further contemplates, that theconcentration of said alcohol is about 15% v/v to about 25% v/v. Thepresent invention further contemplates, that the total concentration ofthe one or more of urea and guanidine thiocyanate (GITC) in said aqueousextraction solution is about 4 M to about 5 M. The present inventionfurther contemplates, that the cellular source material is selected fromliving cellular source material and fixed cellular source material. Thepresent invention further contemplates, that the living cellular sourcematerial comprises a suspension of single cells. The present inventionfurther contemplates, that the suspension of cells comprises bacteria.The present invention further contemplates, that the bacteria areMycobacteria. The present invention further contemplates, that thesuspension of cells comprises yeast. The present invention furthercontemplates, that the aqueous extraction solution is enzyme-free. Thepresent invention further contemplates, that the aqueous extractionsolution is protease-free. The present invention further contemplates,that the fixed cellular source material comprises formalin-fixedparaffin embedded (FFPE) material.

The present invention contemplates a method of inactivating and killingMycobacterium, said method comprising: providing i) cellular sourcematerial comprising Mycobacterium and ii) an aqueous extraction solutioncomprising one or more amine monomers; contacting said cellular sourcematerial with said extraction solution resulting in lysis of thecellular material and the extraction of the nucleic acids. The presentinvention further contemplates, that the amine monomer is a primaryamine monomer. The present invention further contemplates, that theamine monomer is 2,2′-(ethylenedioxy)bis(ethylamine) (EDBE). The presentinvention further contemplates, that the amine monomer is selected fromone or more of 1,3-diaminopropane and 3-amino-1-propanol. The presentinvention further contemplates, that the aqueous extraction solutionfurther comprises a chaotrope. The present invention furthercontemplates, that the chaotrope is selected from one or more of thegroup consisting of urea, guanidine thiocyanate (GITC), ethanol andbutanol. The present invention further contemplates, that the aqueousextraction solution further comprises one or more of a detergent and analcohol. The present invention further contemplates, that the detergentis selected from one or more of the group consisting of Tween™,polysorbates, deoxycholate, sodium deoxycholate and sodium dodecylsulfate (SDS), NP-40 and Triton™ X-100. The present invention furthercontemplates, that the detergent is about 8% v/v to about 15% v/v. Thepresent invention further contemplates, that the alcohol is selectedfrom one or more of the group consisting of ethanol and butanol. Thepresent invention further contemplates, that the concentration of saidalcohol is about 15% v/v to about 25% v/v. The present invention furthercontemplates, that the concentration of amine monomer in said aqueousextraction solution is about 30% v/v to about 50% v/v. The presentinvention further contemplates, that the concentration of the chaotropein said aqueous extraction solution is about 4 M to about 5 M. Thepresent invention further contemplates, that source material comprisingMycobacterium comprises a suspension of single cells. The presentinvention further contemplates, that the aqueous extraction solution isenzyme-free. The present invention further contemplates, that theaqueous extraction solution is protease-free. The present inventionfurther contemplates, that the method further extracts nucleic acid fromsaid Mycobacterium.

The present invention contemplates a method of extracting nucleic acidfrom cellular source material, said method comprising contactingcellular source material with an aqueous extraction solution capable oflysis of the cellular material and extraction of the nucleic acid in asingle step, wherein said aqueous extraction solution comprises anitrogen containing solvent.

The present invention contemplates a method of extracting nucleic acidfrom fixed tissue cellular source material, said method comprisingcontacting cellular source material with an aqueous extraction solutioncapable of lysis of the cellular material and extraction of the nucleicacid in a single step, wherein said aqueous extraction solutioncomprises a nitrogen containing solvent.

The present invention contemplates a method of extracting nucleic acidfrom bacterial cellular source material, said method comprisingcontacting cellular source material with an aqueous extraction solutioncapable of lysis of the cellular material and extraction of the nucleicacid in a single step; wherein said aqueous extraction solutioncomprises a nitrogen containing solvent.

The present invention contemplates a method of extracting nucleic acidfrom yeast cellular source material, said method comprising contactingcellular source material with an aqueous extraction solution capable oflysis of the cellular material and extraction of the nucleic acid in asingle step; wherein said aqueous extraction solution comprises anitrogen containing solvent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows amplification curves from the assays of Example 1.

FIG. 2 shows the results of a one-way ANOVA analysis on the data of FIG.1.

FIG. 3 shows amplification curves from the assays of Example 2.

FIG. 4 shows the results of a one-way ANOVA analysis on the data of FIG.3.

FIG. 5 shows amplification curves from the assays of Example 3.

FIG. 6 shows the results of a one-way ANOVA analysis on the data of FIG.5.

FIGS. 7 (A & B) shows the results of DNA concentration after extractionof yeast from Example 4.

FIG. 8 (A-F) shows amplification curves for the assays of Example 4.

FIG. 9 shows the results of a one-way ANOVA analysis on the data of FIG.8.

FIG. 10 (A-E) shows amplification curves for the assays of Example 5.

FIG. 11 shows the extraction of DNA from 5 micron FFPE curls (notmounted on glass slides) of colorectal cancer (CRC) specimens by Qiagen,Promega and the amine solvent (3A1P) extraction protocol of the presentinvention.

FIG. 12 shows the deltaCt (dCt) of the samples assayed for FIG. 11.

FIG. 13 shows the extraction of DNA from 5 micron FFPE curls (notmounted on glass slides) of lung cancer specimens by Qiagen, Promega andthe amine solvent (3A1P) extraction protocol of the present invention.

FIG. 14 shows the deltaCt (dCt) of the samples assayed for FIG. 13.

FIG. 15 shows the extraction of DNA from 5 micron FFPE curls (notmounted on glass slides) of melanoma specimens by Qiagen, Promega andthe amine solvent (3A1P) extraction protocol of the present invention.

FIG. 16 shows the deltaCt (dCt) of the samples assayed for FIG. 15.

FIG. 17 shows amine solvent extraction protocol used on slide mountedFFPE specimens. The specimens were hepatitis B virus internal control(HBV-IC; Abbott Laboratories, Abbott Park, Ill.). These slides werechosen because the quantity of DNA is a known variable. The HBV ICslides were extracted in LB-EtOH-3A1P isolated with the CSC or incubatedin LB-3A1P with glass slides prior to DNA isolation EtOH was added afterextraction. The figure shows that the glass slides bound a substantialamount of the DNA.

FIG. 18 shows that the addition of magnetic microparticles (MMP) duringthe extraction procedure resulted in recovery that was equivalent torecovery without glass slides.

FIG. 19 shows an equivalent experiment on breast tumor FFPE glassslides.

FIG. 20 shows extraction of 1 to 4 slides demonstrating that recoverycan be increased with the addition of slides to the extractionprocedure.

FIG. 21 shows a one-way ANOVA analysis of the data in FIG. 20.

FIGS. 22 (A & B) shows results of a one-way analysis of the data bysolvent for C. albicans and S. aureus, respectively.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention comprises an aqueous extractionsolution capable of lysing cells and purifying nucleic acid in one step.In this regard, the present invention provides methods and compositionssuitable for the extraction of nucleic acids from cellular sourcematerials using an aqueous or aqueous-base extraction solution(extraction composition), said extraction solution comprising one ormore compounds having at least one nitrogenous group. In a preferredembodiment, the compound is an amine monomer. Aqueous and aqueous-basedare defined as having water as the solvent. However, this does notexclude the inclusion of non-aqueous components providing that they aremiscible in water.

The term “cellular source material” is defined herein to mean anybiological material comprising cells or, in some instances, cell matter(i.e., previously lysed cell constituents). Cellular source material maybe fresh (i.e., not fixed) or may be fixed by methods known to those ofordinary skill in the art. Formalin fixation (and procedures using otheraldehydes) is a common fixation procedure although other methods existand are known to those or ordinary skill in the art. Cellular sourcematerial may also be comprised of one or more tissues.

“Extraction of nucleic acid(s)” shall mean, herein, the release of thenucleic acids from the cellular source material in sufficient quantityfrom other cellular components to the extent that they can be removedfrom the lysate for further processing, if desired. In other words, thenucleic acids are enriched.

“Enriched” or “Enrichment” with regard to the nucleic acids of thepresent invention shall mean that the nucleic acids are at a greaterconcentration relative to the other continuants of the cellular sourcematerial than before the cellular material is subject to the methods andcompositions of the present invention. In other words, the nucleic acidsare “partly purified” or “partly isolated.”

“Purification” or “to Purify” with regard to the nucleic acids of thepresent invention shall mean the removal of continuants of the cellularsource material from a sample. As used herein, the term “purified”refers to nucleic acid sequences that are removed from their naturalenvironment, isolated or separated. “Isolation” and “purification” withregard to the nucleic acids of the present invention shall mean that thenucleic acids are more than 10% free, more than 20% free, more than 30%free, more than 40% free, more than 50% free, more than 60% free, morethan 70% free, more than 80% free, more than 90% free, more than 95%free and more than 99% free from other cellular components with whichthey are naturally associated.

“Single Step” as used herein, is intended to refer to a method ofextracting nucleic acid from cellular source material, whereby the DNAis released from the material and Enriched or Purified, in one step.

In an embodiment of the present invention relating to fixed tissue, thesingle step method described herein eliminates the need for separatede-paraffinization of the materials or an enzymatic digestion of thematerials to release the DNA. In certain embodiments, the DNA iscaptured on a solid support, (e.g., a silica containing surface) withoutthe need for de-paraffinization of the materials or an enzymaticdigestion of the materials to release the DNA. In embodiment of thepresent invention relating to fixed tissue, the single step methoddescribed herein eliminates the need for other lysis methods or anenzymatic digestion of the material. In certain embodiments, the DNA iscaptured on a solid support, (e.g., a silica containing surface) withoutthe need for lysis of the sample to release the DNA from the cellularsource material. In certain embodiments, the yeast DNA is captured on asolid support, (e.g., a silica containing surface) without the need forlysis of the sample to release the DNA from the cellular sourcematerial. In embodiment of the present invention relating to bacterialcellular source material, the single step method described hereineliminates the need for other lysis methods or an enzymatic digestion ofthe material. In certain embodiments, the DNA is released from thematerial and Enriched or Purified, without the need for inactivation ofthe bacteria. In certain other embodiments, the DNA is captured on asolid support, (e.g., a silica containing surface) without the need forlysis of the sample or an enzymatic digestion of the material to releasethe DNA from the cellular source material.

The present invention is not limited to any particular source forcellular source material. The cell source material may be obtained fromany kind of cell or tissue that contains nucleic acid. This includesviruses and cells containing viruses, bacteria (for example, one or moreof Mycobacteria spp., for example, M. tuberculosis) and cells containingbacteria, all other prokaryotic cells, yeast (for example, one or moreof Saccharomyces spp. or Candida spp., e.g., C. albicans), all otherfungus, botanical (i.e., plant) and animal cells, etc. Cellular sourcematerial may be recently obtained and living or may be non-living.Likewise, the cell source material may be samples preserved viapreservation and fixation compounds and techniques known to one ofordinary skill in the art, a brief summary of which can be found below.Formalin fixed, paraffin embedded (FFPE) tissues are especially suitablefor use in the present invention. The methods and compositions of thepresent invention lyse Mycobacteria and other pathogenic organismsthereby killing them and lessening or eliminating danger ofcontamination from the sample.

The present invention does not require any pre-treatment or pre-handlingof fresh (i.e., non-fixed) cellular source material. Further, ifpre-treatment or pre-handling procedures are used, the present inventionis not limited to any particular pre-treatment or pre-extractionhandling procedure. However, in some instances pre-treatment orpre-handling of the cellular source material may be advantageous. Forexample, it may be desirable to concentrate suspended cells bycentrifugation. Also, large tissues (fresh, fixed or fixed and embedded)are easier to handle if processed (e.g., cut or ground) into smallersections. Methods suitable for preprocessing of samples are known in theart and include, but are not limited to, sonication of cells (U.S. Pat.No. 6,686,195) with or without the presence of grinding particles,mixing (e.g., vortexing), high powered agitation with grinding particles(U.S. Pat. No. 5,464,773) (bead beating, ball mills) or the use of highpressure mechanical shearing (e.g., French pressure cell press, as isknown in the art). Further, enzymatic methods that use particularenzymes such as zymolase (Salazar and Asenjo, ibid; U.S. Pat. No.5,688,644) to weaken the cell walls. Further still, if a particular celltype is being targeted, isolation of that particular cell type from alarger population may be preferred. However, these procedures aresuggested for ease of handling and expedience and not because thepresent invention requires pre-treatment or pre-handling.

In an embodiment of the present invention, the present invention uses anextraction solution (composition) comprising one or more amine monomers.Although the present invention is not limited to any particular theory,in the context of the present invention it is believed that the reagenthaving one or more amine monomers functions as a solvent. Examples ofreagents comprising one or more amine monomers are, for example, include2,2′-ethylenedioxy)bis(ethylamine): C₆H₁₆N₂O₂) (EDBE). EDBE is a primaryamine monomer. Further, the present invention is not limited to anyparticular amine monomer or primary amine monomer. For example, theprimary amine monomer diaminopropane (e.g., 1,2-diaminopropane and1,3-diaminopropane) is also useful in the present invention, asexemplified below. Further, 3-amino-1-propanol (3A1P) is useful in thepresent invention, as exemplified below, and exhibits lower toxicitythan the other two amine monomers named above. The final concentrationof the amine monomer in the extraction solution of the present inventionis about 15% to about 50% or about 20% to about 45% or about 40%.Preliminary results suggest that ammonium hydroxide (NH₄OH) is effectiveas a substitute for amine monomers, albeit with a reduced effectiveness.

With regard to the present invention, a “Primary Amine” is defined as anamine wherein only one of the hydrogen atoms in the ammonia molecule hasbeen replaced. That means that the formula of the primary amine will beRNH₂. A secondary amine is defined as an amine wherein two of thehydrogens atoms in the ammonia molecule have been replaced. That meansthat the formula of the primary amine will be RNHR. A “Tertiary Amine”is defined as an amine wherein three of the hydrogens atoms in theammonia molecule have been replaced. An “Amine Monomer” is a compoundhaving one or more amine groups.

In other embodiments, it is contemplated that combinations of reagentsmay be used in the aqueous extraction solutions of the present inventionwith suitable results. For example, the amine monomers discussed above(one or more of 2,2′-eththylenedioxy)bis(ethylamine) (EDBE),1,3-diaminopropane (DAP), 2-amino-1-butanol (AB),2-(2-aminoethoxy)ethanol (AEE), 2-amino-6-methylheptane (AMH),2-amino-2-methyl-1-propanol (AMP), amino-2-propanol (A2P),1,5-diamino-2-methylpentane (DMP) and 3-amino-1-propanol (3A1P)) may beused with urea and/or GITC and/or NH₄OH. One of ordinary skill in theart is able to determine acceptable concentrations and conditions withonly routine experimentation.

In another embodiment, it is contemplated that aqueous extractionsolutions of the present invention may comprise urea and/or GITC withoutthe addition of an amine monomer. Again, one of ordinary skill in theart will be able to determine acceptable concentrations and conditionswith only routine experimentation.

The extraction solution (composition) of the present invention may also,optionally, comprise other organic solvents such as, for example,dimethyl sulfoxide (DMSO), alcohols and limonene. The finalconcentration of the organic solvent in the extraction composition ofthe present invention, if present, is about 10% to 30%, about 15% toabout 25% or about 20%.

The extraction solution (composition) of the present invention may alsocomprise a chaotropic agent such as, for example, urea, guanidinethiocyanate (GITC), ethanol or butanol. Others are known to those ofskill in the art. A chaotropic agent is a substance that disrupts thestructure of and denatures macromolecules such as proteins. Chaotropicagents act by interfering with the intermolecular interactions mediatedby non-covalent force such as hydrogen bonds. The final concentration ofthe chaotropic agent in the extraction composition of the presentinvention is about 3.0 M to about 6.0 M, about 4.0 M to about 5.0 M orabout 4.7 M.

Further, the extraction solution (composition) of the present inventioncomprises one or more detergents. Detergents are characterized byhydrophilic head and a hydrophobic tail. Detergents are typically usedin cell and tissue work. Non-ionic detergents are preferred. Detergentssuitable for use in the present invention include, but are not limitedto, Tween™, polysorbates, deoxycholate, sodium deoxycholate and sodiumdodecyl sulfate (SDS), NP-40 and Triton™ X-100 (Sigma-Aldrich, St.Louis, Mo.). The final concentration of the detergent in the extractioncomposition of the present invention is about 1% to 15%, about 8% toabout 15% or about 10%. Although the present invention is not limited bytheory, it is generally thought that moderate concentrations of mild(i.e., non-ionic) detergents compromise the integrity of cell membranes,thereby facilitating lysis of cells and extraction of solublecomponents.

The pH of the lysis solution of the present invention is above about 7.The pH of the lysis solution of the present invention may be as high asabout pH 10-13 or 12-13. The pH may be adjusted and maintained byselection of the components of the extraction composition of the presentinvention or by the use of buffers. The use of buffers is well known toone of ordinary skill in the art. An exemplary buffer is Tris-HCL.

Further, unlike prior art methods, the present method can be performedwithout the use of enzymes (e.g., proteases) for the breakdown of, forexample, tissues, although the use of enzymes is not contraindicated.

The mixture may optionally be warmed or heated to aid in the release ofthe nucleic acids. The temperature used may range from about 70° C. toabout 90° C. and about 80° C. to about 90° C. and temperatures of about85° C.

The present invention is suitable for use on fixed cells and tissues.Non-limiting examples of fixatives and fixation procedures include, forexample, crosslinking fixatives (e.g., aldehydes, such asglutaraldehyde, formaldehyde (formalin), etc.). Crosslinking fixativesact by creating covalent chemical bonds between proteins in tissue.These crosslinking fixatives, especially formaldehyde, tend to preservethe secondary structure of proteins and may protect significant tertiarystructure as well. Precipitating (or denaturing) fixatives such asmethanol, ethanol, acetic acid and acetone are also known.

The oxidizing fixatives can react with various side chains of proteinsand other biomolecules, allowing formation of crosslinks that stabilizetissue structure. Osmium tetroxide, potassium dichromate, chromic acid,and potassium permanganate all find use in certain specific histologicalpreparations.

Hepes-glutamic acid buffer-mediated organic solvent protection effect(HOPE) gives formalin-like morphology, excellent preservation of proteinantigens for immunohistochemistry and enzyme histochemistry, good RNAand DNA yields and absence of crosslinking proteins.

Fixed cellular source material sample such as tissue and cell samplesare often embedded to preserve over all structure and to provide supportfor further processing. Traditionally, such samples have been embeddedin paraffin. Fixed cellular source material samples are typically fixedin, for example, formalin prior to paraffin embedment creatingformalin-fixed, paraffin embedded (FFPE) samples. Procedures for thefixation of cells and tissues and embedment of cells and tissues areknown to one of ordinary skill in the art (see, e.g., Leeson and Leeson,Histology, 1981, W. B. Saunders Co., pages 6-8 and on the World Wide Webat en.wikipedia.org/wiki/Histology#Embedding). In the prior art, nucleicacid extraction from FFPE cell source material has been accomplishedonly by using difficult, multistep, time consuming procedures. Thecompositions and procedures of the present invention are directedtowards a simplified, efficient procedure.

The present invention is directed towards a new and non-obvious processthat eliminates the need for the deparaffinization and proteasedigestion steps of tissues for the extraction, enrichment, isolationand/or purification of nucleic acids. A single aqueous-based solution isused for the extraction and binding of the nucleic acids to a solidmatrix (if binding is desired). The organic solvents contained in thesolution are completely miscible with no phase separation of the organicsolvents. The FFPE tissue is mixed with the solution, the tissue isdisrupted, nucleic acids are released and the nucleic acids are capturedon, for example, a solid matrix such as a silica containing solid matrixor are removed from the solution by any other method known to those ofordinary skill in the art. The matrix may be particles and may bemagnetic particles. After the nucleic acids are captured on the solidmatrix, the process uses simple wash steps and elution of the nucleicacids from the matrix for the final purification or use. Extractednucleic acid can be further purified, if desired, by methods known toone of ordinary skill in the art.

Extracted nucleic acid can be used by any procedures known to those ofordinary skill in the art. Examples of such uses include hybridizationassays (northern blots, Southern blots, etc), amplification assays (see,for example, U.S. Pat. No. 4,683,195), sequencing, copying,incorporation into expression vectors or any useful combination thereof.Further, the compositions and methods of the present invention aresuitable for use on samples previously used for FISH (fluorescent insitu hybridization) analysis or other protocol wherein the nucleic acidis not destroyed.

All citations (patents, patent application publications, journalarticles, textbooks, and other publications) mentioned in thespecification are indicative of the level of skill of those in the artto which the disclosure pertains. All such publications are incorporatedherein by reference to the same extent as if each individual publicationwere specifically and individually indicated to be incorporated byreference.

The invention illustratively described herein may be suitably practicedin the absence of any element(s) or limitation(s), which is/are notspecifically disclosed herein. Thus, for example, each instance hereinof any of the terms “comprising,” “consisting essentially of,” and“consisting of” may be replaced with either of the other two terms.Likewise, the singular forms “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. Thus, forexample, references to “the method” includes one or more methods and/orsteps of the type, which are described herein and/or which will becomeapparent to those ordinarily skilled in the art upon reading thedisclosure.

Embodiments of Extracting Nucleic Acid from Cellular Source Material ina Single Step

In one embodiment, the present invention is directed to a method ofextracting nucleic acid from cellular source material, said methodcomprising contacting cellular source material with an aqueousextraction solution capable of lysis of the cellular material andextraction of the nucleic acid in a single step. In one embodiment, theaqueous extraction solution comprises a nitrogen containing solventselected from one or more amine monomers and one or more amides. In oneembodiment, the aqueous extraction solution comprises an amine monomerand an amide. In another embodiment, the aqueous extraction solutioncomprises a primary amine monomer. In one embodiment, the amine monomeris 2,2′-(ethylenedioxy)bis(ethylamine) (EDBE). In one embodiment, theamine monomer is 1,3-diaminopropane. In one embodiment, the animemonomer is 3-amino-1-propanol. In one embodiment two or more aminemonomers are used concurrently. In one embodiment, the amine monomer isselected from one or more of 2,2′-eththylenedioxy)bis(ethylamine)(EDBE), 1,3-diaminopropane (DAP), 2-amino-1-butanol (AB),2-(2-aminoethoxy)ethanol (AEE), 2-amino-6-methylheptane (AMH),2-amino-2-methyl-1-propanol (AMP), amino-2-propanol (A2P),1,5-diamino-2-methylpentane (DMP) and 3-amino-1-propanol (3A1P). In oneembodiment, the concentration of the amine monomer in the aqueousextraction solution is about 15% to about 50%. In one embodiment, theconcentration of the amine monomer in the aqueous extraction solution isabout 20% to about 45%. In one embodiment, the concentration of theamine monomer in the aqueous extraction solution is about 45% to about50%. In one embodiment, the aqueous extraction solution furthercomprises a chaotrope. In one embodiment, the chaotrope is selected fromthe group consisting of urea, guanidine thiocyanate (GITC), ethanol andbutanol. In one embodiment, the chaotrope in said aqueous extractionsolution is about 4M to about 5M. In one embodiment, the chaotrope insaid aqueous extraction solution is about 4.5M to about 5M. In oneembodiment, the aqueous extraction solution further comprises one ormore of a detergent and an alcohol. Suitable detergents include Tween™,polysorbates, deoxycholate, sodium deoxycholate and sodium dodecylsulfate (SDS), NP-40 and Triton™ X-100. Suitable alcohols includeethanol, butanol. In one embodiment, the concentration of the detergentis between about 1% to about 15% or about 8% to about 15%. In oneembodiment, the concentration of the detergent is between about 10% toabout 15%. In one embodiment, the concentration of the detergent isbetween about 12% to about 15%. In one embodiment, the concentration ofthe alcohol is about 15% to about 25%. In one embodiment, theconcentration of the alcohol is about 10% to about 40%. In oneembodiment, the concentration of the alcohol is about 20% to about 35%.In one embodiment, the concentration of the alcohol is about 20% toabout 25%. In one embodiment, the concentration of the alcohol is about23% to about 25%. In another embodiment, the cellular source material isselected from the group consisting of tissue, animal tissue, mammaliantissue, human tissue, human tumor tissue, human tissue containingviruses, fixed human tissue, animal cells, mammalian cells, human cells,human cells containing viruses, bacteria, mycobacteria, fungus, yeast,and plant tissue or plant cells, blood containing cells, sputumcontaining cells.

Embodiments of Extracting Nucleic Acid from Fixed Tissue Cellular SourceMaterial in a Single Step

In one embodiment, the present invention is directed to a method ofextracting nucleic acid from Fixed Tissue cellular source material, saidmethod comprising contacting cellular source material with an aqueousextraction solution capable of lysis of the cellular material andextraction of the nucleic acid in a single step. In one embodiment, thefixed cellular source material is formalin-fixed paraffin embeddedtissue. In one embodiment, the aqueous extraction solution comprises anitrogen containing solvent selected from one or more amine monomers andone or more amides. In one embodiment, the aqueous extraction solutioncomprises an amine monomer and an amide. In another embodiment, theaqueous extraction solution comprises a primary amine monomer. In oneembodiment, the amine monomer is 2,2′-(ethylenedioxy)bis(ethylamine)(EDBE). In one embodiment, the amine monomer is 1,3-diaminopropane. Inone embodiment, the anime monomer is 3-amino-1-propanol. In oneembodiment two or more amine monomers are used concurrently. In oneembodiment, the amine monomer is selected from one or more of2,2′-eththylenedioxy)bis(ethylamine) (EDBE), 1,3-diaminopropane (DAP),2-amino-1-butanol (AB), 2-(2-aminoethoxy)ethanol (AEE),2-amino-6-methylheptane (AMH), 2-amino-2-methyl-1-propanol (AMP),amino-2-propanol (A2P), 1,5-diamino-2-methylpentane (DMP) and3-amino-1-propanol (3A1P). In one embodiment, the concentration of theamine monomer in the aqueous extraction solution is about 15% to about50%. In one embodiment, the concentration of the amine monomer in theaqueous extraction solution is about 20% to about 45%. In oneembodiment, the concentration of the amine monomer in the aqueousextraction solution is about 45% to about 50%. In one embodiment, theaqueous extraction solution further comprises a chaotrope. In oneembodiment, the chaotrope is selected from the group consisting of urea,guanidine thiocyanate (GITC), ethanol and butanol. In one embodiment,the chaotrope in said aqueous extraction solution is about 4M to about5M. In one embodiment, the chaotrope in said aqueous extraction solutionis about 4.5M to about 5M. In one embodiment, the aqueous extractionsolution further comprises one or more of a detergent and an alcohol.Suitable detergents include Tween™, polysorbates, deoxycholate, sodiumdeoxycholate and sodium dodecyl sulfate (SDS), NP-40 and Triton™ X-100.Suitable alcohols include ethanol, butanol. In one embodiment, theconcentration of the detergent is between about 1% to about 15% or about8% to about 15%. In one embodiment, the concentration of the detergentis between about 10% to about 15%. In one embodiment, the concentrationof the detergent is between about 10% to about 40%. In one embodiment,the concentration of the alcohol is about 20% to about 35%. In oneembodiment, the concentration of the alcohol is about 20% to about 25%.In one embodiment, the concentration of the alcohol is about 23% toabout 25%.

Embodiments of Extracting Nucleic Acid from Bacterial Cellular SourceMaterial in a Single Step

In one embodiment, the present invention is directed to a method ofextracting nucleic acid from bacterial cellular source material, saidmethod comprising contacting cellular source material with an aqueousextraction solution capable of lysis of the cellular material andextraction of the nucleic acid in a single step. In one embodiment, thebacterial cellular source material is mycobacterial cells. In anotherembodiment, the bacterial cellular source material is MycobacteriumTuberculosis. In another embodiment, the bacteria are found in humansputum. In another embodiment, the bacteria is inactivated in the singlestep. In one embodiment, the aqueous extraction solution comprises anitrogen containing solvent selected from one or more amine monomers andone or more amides. In one embodiment, the aqueous extraction solutioncomprises an amine monomer and an amide. In another embodiment, theaqueous extraction solution comprises a primary amine monomer. In oneembodiment, the amine monomer is 2,2′-(ethylenedioxy)bis(ethylamine)(EDBE). In one embodiment, the amine monomer is 1,3-diaminopropane. Inone embodiment, the anime monomer is 3-amino-1-propanol. In oneembodiment two or more amine monomers are used concurrently. In oneembodiment, the amine monomer is selected from one or more of2,2′-eththylenedioxy)bis(ethylamine) (EDBE), 1,3-diaminopropane (DAP),2-amino-1-butanol (AB), 2-(2-aminoethoxy)ethanol (AEE),2-amino-6-methylheptane (AMH), 2-amino-2-methyl-1-propanol (AMP),amino-2-propanol (A2P), 1,5-diamino-2-methylpentane (DMP) and3-amino-1-propanol (3A1P) In one embodiment, the concentration of theamine monomer in the aqueous extraction solution is about 15% to about50%. In one embodiment, the concentration of the amine monomer in theaqueous extraction solution is about 20% to about 45%. In oneembodiment, the concentration of the amine monomer in the aqueousextraction solution is about 45% to about 50%. In one embodiment, theaqueous extraction solution further comprises a chaotrope. In oneembodiment, the chaotrope is selected from the group consisting of urea,guanidine thiocyanate (GITC), ethanol and butanol. In one embodiment,the chaotrope in said aqueous extraction solution is about 4M to about5M. In one embodiment, the chaotrope in said aqueous extraction solutionis about 4.5M to about 5M. In one embodiment, the aqueous extractionsolution further comprises one or more of a detergent and an alcohol.Suitable detergents include Tween™, polysorbates, deoxycholate, sodiumdeoxycholate and sodium dodecyl sulfate (SDS), NP-40 and Triton™ X-100.Suitable alcohols include ethanol, butanol. In one embodiment, theconcentration of the detergent is between about 1% to about 15% or about8% to about 15%. In one embodiment, the concentration of the detergentis between about 10% to about 15%. In one embodiment, the concentrationof the detergent is between about 10% to about 40%. In one embodiment,the concentration of the alcohol is about 20% to about 35%. In oneembodiment, the concentration of the alcohol is about 20% to about 25%.In one embodiment, the concentration of the alcohol is about 23% toabout 25%.

Embodiments of Extracting Nucleic Acid from Yeast Cellular SourceMaterial in a Single Step

In one embodiment, the present invention is directed to a method ofextracting nucleic acid from Yeast cellular source material, said methodcomprising contacting cellular source material with an aqueousextraction solution capable of lysis of the cellular material andextraction of the nucleic acid in a single step. In one embodiment, theaqueous extraction solution comprises a nitrogen containing solventselected from one or more amine monomers and one or more amides. In oneembodiment, the aqueous extraction solution comprises an amine monomerand an amide. In another embodiment, the aqueous extraction solutioncomprises a primary amine monomer. In one embodiment, the amine monomeris 2,2′-(ethylenedioxy)bis(ethylamine) (EDBE). In one embodiment, theamine monomer is 1,3-diaminopropane. In one embodiment, the animemonomer is 3-amino-1-propanol. In one embodiment two or more aminemonomers are used concurrently. In one embodiment, the amine monomer isselected from one or more of 2,2′-eththylenedioxy)bis(ethylamine)(EDBE), 1,3-diaminopropane (DAP), 2-amino-1-butanol (AB),2-(2-aminoethoxy)ethanol (AEE), 2-amino-6-methylheptane (AMH),2-amino-2-methyl-1-propanol (AMP), amino-2-propanol (A2P),1,5-diamino-2-methylpentane (DMP) and 3-amino-1-propanol (3A1P). In oneembodiment, the concentration of the amine monomer in the aqueousextraction solution is about 15% to about 50%. In one embodiment, theconcentration of the amine monomer in the aqueous extraction solution isabout 20% to about 45%. In one embodiment, the concentration of theamine monomer in the aqueous extraction solution is about 45% to about50%. In one embodiment, the aqueous extraction solution furthercomprises a chaotrope. In one embodiment, the chaotrope is selected fromthe group consisting of urea, guanidine thiocyanate (GITC), ethanol andbutanol. In one embodiment, the chaotrope in said aqueous extractionsolution is about 4M to about 5M. In one embodiment, the chaotrope insaid aqueous extraction solution is about 4.5M to about 5M. In oneembodiment, the aqueous extraction solution further comprises one ormore of a detergent and an alcohol. Suitable detergents include Tween™,polysorbates, deoxycholate, sodium deoxycholate and sodium dodecylsulfate (SDS), NP-40 and Triton™ X-100. Suitable alcohols includeethanol, butanol. In one embodiment, the concentration of the detergentis between about 1% to about 15% or about 8% to about 15%. In oneembodiment, the concentration of the detergent is between about 10% toabout 15%. In one embodiment, the concentration of the detergent isbetween about 10% to about 40%. In one embodiment, the concentration ofthe alcohol is about 20% to about 35%. In one embodiment, theconcentration of the alcohol is about 20% to about 25%. In oneembodiment, the concentration of the alcohol is about 23% to about 25%.

Embodiments of Extracting Nucleic Acid from FISH-Assayed Cellular SourceMaterial in a Single Step

In one embodiment, the present invention is directed to a method ofextracting nucleic acid from FISH-assayed cellular source material, saidmethod comprising contacting cellular source material with an aqueousextraction solution capable of lysis of the cellular material andextraction of the nucleic acid in a single step. In one embodiment, theaqueous extraction solution comprises a nitrogen containing solventselected from one or more amine monomers and one or more amides. In oneembodiment, the aqueous extraction solution comprises an amine monomerand an amide. In another embodiment, the aqueous extraction solutioncomprises a primary amine monomer. In one embodiment, the amine monomeris 2,2′-(ethylenedioxy)bis(ethylamine) (EDBE). In one embodiment, theamine monomer is 1,3-diaminopropane. In one embodiment, the animemonomer is 3-amino-1-propanol. In one embodiment, the amine monomer isselected from one or more of 2,2′-eththylenedioxy)bis(ethylamine)(EDBE), 1,3-diaminopropane (DAP), 2-amino-1-butanol (AB),2-(2-aminoethoxy)ethanol (AEE), 2-amino-6-methylheptane (AMH),2-amino-2-methyl-1-propanol (AMP), amino-2-propanol (A2P),1,5-diamino-2-methylpentane (DMP) and 3-amino-1-propanol (3A1P). In oneembodiment two or more amine monomers are used concurrently. In oneembodiment, the concentration of the amine monomer in the aqueousextraction solution is about 15% to about 50%. In one embodiment, theconcentration of the amine monomer in the aqueous extraction solution isabout 20% to about 45%. In one embodiment, the concentration of theamine monomer in the aqueous extraction solution is about 45% to about50%. In one embodiment, the aqueous extraction solution furthercomprises a chaotrope. In one embodiment, the chaotrope is selected fromthe group consisting of urea, guanidine thiocyanate (GITC), ethanol andbutanol. In one embodiment, the chaotrope in said aqueous extractionsolution is about 4M to about 5M. In one embodiment, the chaotrope insaid aqueous extraction solution is about 4.5M to about 5M. In oneembodiment, the aqueous extraction solution further comprises one ormore of a detergent and an alcohol. Suitable detergents include Tween™,polysorbates, deoxycholate, sodium deoxycholate and sodium dodecylsulfate (SDS), NP-40 and Triton™ X-100. Suitable alcohols includeethanol, butanol. In one embodiment, the concentration of the detergentis between about 1% to about 15% or about 8% to about 15%. In oneembodiment, the concentration of the detergent is between about 10% toabout 15%. In one embodiment, the concentration of the detergent isbetween about 10% to about 40%. In one embodiment, the concentration ofthe alcohol is about 20% to about 35%. In one embodiment, theconcentration of the alcohol is about 20% to about 25%. In oneembodiment, the concentration of the alcohol is about 23% to about 25%.

The terms and expressions, which have been employed, are used as termsof description and not of limitation. In this regard, where certainterms are defined, described, or discussed herein, all such definitions,descriptions, and discussions are intended to be attributed to suchterms. There also is no intention in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof.

It is recognized that various modifications are possible within thescope of the claimed invention. Thus, it should be understood that,although the present invention has been specifically disclosed in thecontext of preferred embodiments and optional features, those skilled inthe art may resort to modifications and variations of the conceptsdisclosed herein. Such modifications and variations are considered to bewithin the scope of the invention as defined by the appended claims.

EXEMPLIFICATION Example 1

The concept of the present invention is that nucleic acids can be easilyenriched, purified from or isolated from, for example, formaldehydefixed paraffin embedded (FFPE) tissue using a single step lysis bufferthat will allow the DNA to be released from the sample and captured on asolid support, (e.g., a silica containing surface) or otherwise enrichedor isolated, without the need for de-paraffinization of the sample noran enzymatic digestion of the sample to release the DNA from thecellular source material, e.g., fixed tissue. One of ordinary kill inthe art will understand that the procedures of the present invention arealso suitable for the extraction, purification, isolation and enrichmentof nucleic acids from samples that are not FFPE, such as, but notlimited to, bacteria, yeast, tissues, etc.

The basic lysis buffer (LB) used in the extractions contains 4.7 Mguanidine thiocyanate (GITC), 10% Tween-20 and 100 mM tris buffer, pH7.8. The lysis-ethanol (LB-EtOH) solution was made using 70 ml of thelysis buffer and adding 35 ml of 95% ethanol. The FFPE extractionsolution containing 2,2′-(ethylenedioxy)bis(ethylamine) (EDBE, CASNumber 929-59-9) was made by mixing 9 ml of the LB-EtOH solution with 6ml of EDBE for a 40% EDBE solution in LB-EtOH (LB-EtOH-EDBE). The Wash 1solution for all the samples is the LB-EtOH solution. The Wash 2solution for all the samples is 70% ethanol and water. The elutionsolution is water. The silica coated magnetic microparticles (MMP) usedin the protocol are Abbott Laboratories mMicroparticlesDNA code itemMD205A although equivalent particles are commercially available (e.g.,Promega Corp., Madison, Wis.; Life Technologies, Grand Isle, N.Y.; BangsLaboratories, Fishers, Ind.).

The sample extractions were done using a Promega Maxwell extractor. Thismethod transfers magnetic particles between chambers in a cartridgecontaining various solutions used in the extraction protocol. Theextraction protocol involves magnetic particles being transferred fromone chamber to another. The transfer is done by capturing the magneticparticles in a chamber on the surface of a plunger into which a magneticrod has been inserted. The plunger is then moved to a different chamberand the particles are released from the surface of the plunger by movingthe magnetic rod out of the plunger. The plunger without the magneticrod can be used to mix the fluid in the chamber by an up and downmovement in the fluid. In the protocol used for the FFPE extraction, thelysate and particle incubation and the washes were performed at roomtemperature. The elution step was performed in a separate elution tubethat is heated to 70° C. The extraction cartridge had seven chambers.The first chamber was used for the FFPE lysates solution and the otherchambers were used to hold magnetic particles or wash solutions. Chamber2 contained 200 microliters (μl) of LB-EtOH and 25 microliters of MMP.Chamber 3 contained 800 microliters of Wash 1. Chambers 4, 5 and 6contained 900 microliters of Wash 2. Chamber 7 was empty. The elutiontube contained 100 microliters of water. The protocol first transferredthe MMPs from chamber 2 to chamber 1 containing the FFPE lysatessolution. The FFPE lysates solution was mixed with the magneticparticles for ten minutes. All the wash steps were mixed for one minute.The elution step was an incubation for ten minutes with mixing.

Sample material consisted of a FFPE thyroid tissue block sectioned into5 micron sections with single paraffin containing sections placed into 2ml snap cap polypropylene microcentrifuge tubes. The sections weresequentially numbered in the tubes.

Ten sequential sections were extracted in the following manner. To eachsection either 1.5 ml of LB-EtOH or 1.5 ml LB-EtOH-EDBE was added suchthat every other section contained the same lysis buffer solution. Theodd numbered samples contained LB-EtOH and the even numbered samplescontained LB-EtOH-EDBE. In this manner, any differences in the paraffinsection were minimized. All the samples were then incubated at 78° C.for four hours in a stationary temperature controlled heating blockwithout mixing. After the heating step was completed, the lysates weredirectly added to chamber 1 of the Promega Maxwell extraction cartridgesand extracted as described above.

The eluates from the extraction were analyzed using a PCR assay forhuman genomic DNA. PCR is well known to those of ordinary skill in theart. This assay detects the presence of exon 13 of the BRAF gene. Thisgene encodes a protein called B-Raf which is involved in directing cellgrowth. The PCR assay was used to measure the relative amount of DNAisolated from the samples. In the assay, the signal generated by afluorescent probe increases with each heating-cooling cycle in the PCRamplification. The more DNA in the original sample, the earlier thesignal is detected. The cycle at which the signal is detected is calledthe cycle threshold (CT). A sample with twice the amount of genomic DNAthan another sample will have a CT value 1 CT lower than the othersample. A sample with four times the amount of DNA than another samplewill have a CT value 2 CTs lower than the other sample. The CT values ofthe extracts was determined using this method. Two replicate assays weredone for each sample. The amplification curves from the assays are shownin FIG. 1. FIG. 1 shows a clear difference between the samples extractedwith the LB-EtOH lysis buffer and the LB-EtOH-EDBE lysis buffer. Acalculation of the cycle thresholds shows that there is over a 2 CTdifference between the two lysis buffers which translates to over afour-fold increase in the amount of DNA extracted with the EDBEcontaining lysis buffer. FIG. 2 shows a One-way means ANOVA test on thedata presented in FIG. 1.

Means for One-Way Anova of FIG. 2

Lower Upper Level Number Mean Std Error 95% 95% LB-EtOH 10 28.85200.21190 28.407 29.297 LB-EtOH-EDBE 10 26.5170 0.21190 26.072 26.962

Example 2

The concept of the present invention was further explored using a secondsolvent, 1,3-diaminopropane. The extraction was performed as describedabove although only two replicate samples were done with each lysisbuffer. The first lysis buffer was the LB-EtOH buffer (referred to as“LB-EtOH” in the table directly below) and the second was LB-EtOHcontaining 20% 1,3-diaminopropane (DP, CAS number 109-76-2)) (referredto as “diaminopropane” in the table directly below). The FFPE sectionswere from the same tissue sample as used above. The incubation,extraction, and assay conditions were the same as described above. Theamplification curves from the assay are shown below in FIG. 3. FIG. 3shows a clear difference between the samples extracted with the LB-EtOHlysis buffer and the LB-EtOH-diaminopropane lysis buffer. A calculationof the cycle thresholds shows that there is over a 2 CT differencebetween the two lysis buffers which translates to over a four-foldincrease in the amount of DNA extracted with the 1,3-diaminopropanecontaining lysis buffer. FIG. 4 shows a One-way means ANOVA test on thedata presented in FIG. 3.

Means for One-Way Anova for FIG. 4

Lower Upper Level Number Mean Std Error 95% 95% diaminopropane 2 26.00000.23033 25.009 26.991 LB-EtOH 2 28.2400 0.23033 27.249 29.231

Example 3

The present invention was further explored using the addition ofammonium hydroxide to the lysis buffer to determine if the amine groupspresent on the solvents used above influence the extraction of DNA fromFFPE samples. The extractions were performed as described above but thesecond lysis buffer tested contained approximately 0.6% ammoniumhydroxide (NH₄OH). This was made by adding 200 microliters ofconcentrated ammonium hydroxide (28 to 30%) to 10 ml of LB-EtOH. Fivereplicate samples containing the same sample material described abovewere used with each extraction buffer. The eluates were assayed asduplicates with the BRAF assay as described above. The amplificationcurves from the assay are shown below in FIG. 5. FIG. 5 shows adifference between the samples extracted with the LB-EtOH lysis bufferand the LB-EtOH—NH₄OH buffer. A calculation of the cycle thresholdsshows that there is over a 1.4 CT difference between the two lysisbuffers which translates to over a two-fold increase in the amount ofDNA extracted with the NH₄OH containing lysis buffer. FIG. 6 shows aOne-way means ANOVA test on the data presented in FIG. 5. While theincrease in DNA extracted with the lysis buffer containing NH₄OH doesnot appear to be as great as that extracted with the other two solvents,it does show that the presence of ammonium ions or amine groups isimportant in the extraction of DNA from FFPE samples.

Means for Oneway Anova for FIG. 5

Lower Upper Level Number Mean Std Error 95% 95% LB-EtOH 10 30.00000.27787 29.416 30.584 NH₄OH 10 28.5970 0.27787 28.013 29.181

Example 4 Example C. albicans Extraction from Whole Blood

The present invention was further explored for the extraction of nucleicacids from yeast from whole blood. This method was compared to thestandard extraction method for yeast from whole blood that uses beadbeating to lyse the yeast.

The sample used was C. albicans at 200 colony forming units permilliliter of human whole blood. The lysis buffer and other reagentsused in the extraction are described in Example 1. The LB-EtOH-EDBEsolution contained 20% EDBE and was made by mixing 15 ml of EDBE with 60ml of LB-EtOH.

The EDBE extractions were performed by adding 1.25 ml of the sample to3.75 LB-EtOH-EDBE and incubating at 80° C. for 45, 60, 75, and 90minutes. The extractions were given a staggered start so that all theincubations finished at the same time. Four samples of each conditionwere processed. Four samples were also incubated with LB-EtOH (no addedEDBE) for 90 minutes.

The bead beating of the sample was done using an Abbott PIexIDBB set atthree bead beating 90 second cycles for at a 6200 speed. Each samplecontained 1.25 ml sample, 150 microliters of lysis buffer (withoutethanol) and approximately 950 milligrams of Zirconia/Yttrium BeadsGlenn Mills (Clifton, N.J.) #7361-00010. After bead beating the tubeswere centrifuged in a Beckman 22R centrifuge 3 minutes at 14,000 rpm.The total volume of supernatant was then extracted along with the EDBEtreated lysates.

The extractions were performed using a PlexIDsp extractor with aprotocol that has a room temperature incubation of the lysates withsilica coated magnetic particles. The extractor uses 24 well plates witheach plate containing a separate reagent for the extraction. Thereagents are described in Example 1. The binding step was for 15 minutesat room temperature with 125 microliters of the magnetic particles inthe wells. The wells containing the bead beating lysate contained 125microliters of magnetic particles plus 1.5 ml of LB-EtOH while the EDBElysates only had the magnetic particles with no additional reagents. Theprotocol used a single Wash 1 plate with 2 ml of LB-EtOH and three Wash2 plates with 2 ml of 70% ethanol. The elution plate contained 300microliters of water for elution. The elution step was at 70° C. for 10minutes.

The DNA content of the samples was measured using a Nanodrop Lite(Thermo Scientific, Wilmington, Del.). See, FIG. 7 A. There may be lowernucleic acid in the EDBE treated samples than the bead beating. Thesample without EDBE or beadbeating gives a lower yield. The bead beatingprotocol eliminates a great deal of protein from the solution and thenucleic acid extraction appears to be more efficient with the beadbeating step. The A260/A280 ratio is higher with the EDBE samples. See,FIG. 7B.

Assay Eluates. Set Up Assays as Above.

C. albicans assay. for 30 assays 1) Primer IDT #42562400 0.075 ul/rx2.25 ul 2) Primer IDT #42562401 0.075 ul/rx 2.25 ul 3) Probe 186591515-10.5 ul/rx 1.5 ul 4) 2X Taqman Buffer AB #4324018 12.5 ul/rx 375 ul 5)10X IPC mix AB #4308332 2.5 ul/rx 75 ul 6) 50X IPC template AB #43046620.5 ul/rx 15 ul 7) Water. (MD203A-elution buffer) 4.3 ul/rx 129 ul Makemaster mix and add 20 ul to each well in plate 8) Sample 5.0 ul/rx eachseparate Put the Samples at −20 C. when done. load 24 positions- thenadd 5 ul of sample.

Position in Cycler

#1 #2 #3 A 1 9 17 B 2 10 18 C 3 11 19 D 3 12 20 E 5 13 21 F 6 14 22 G 715 23 H 8 16 24 G 7 15 23 H 8 16 24

Taqman buffer is Applied Biosystems (Life Technologies, Grand Island,N.Y.) Universal PCR Master Mix, part #4324018. IPC mix (#408332) and IPCtemplate (#4304662) are exogenous internal positive controls fromApplied Biosystems. Used program ibisQPCR (ngul) LDA in cycler AM01789in B132 to amplify the nucleic acid. Load amplification intoMultianalyse 4. FIG. 8A combines results from FIGS. 8B-F. FIG. 8B showsresults of bead beat and 90 minute with no EDBE. FIG. 8C shows resultsof bead beat, 45 minute EDBE and 90 minute no EDBE. FIG. 8D showsresults of bead beat, 60 minute EDBE and 90 minute no EDBE. FIG. 8Eshows results of bead beat, 75 minute EDBE and 90 minute no EDBE. FIG.8F shows results of bead beat, 90 minute EDBE and 90 minute no EDBE.

FIG. 9 shows Ct results after amplification of nucleic acids. 75 and 90minute incubations with EDBE were as effective as the prior arttechnique incorporating bead beating. The sample without EDBE did notextract the yeast sample well with over 10 fold less yeast DNA in thesample.

Example 5

EDBE Extraction of M. tuberculosis (MTB) from Sputum.

Sputum samples were used to test the ability of the LB-EtOH-EDBEsolution to lyse and extract nucleic acid from MTB. Three sputum sampleswere aliquoted into tared 15 ml polypropylene tubes as follows. A 5 mlpipette with the conical end removed was used to transfer the sputum.The volume of sputum in the tube was calculated and a heat killed MTBculture was then added to the sample at 3000 cfu/ml, with 12.3microliters added per ml of sputum.

specimen tube tare total sample A 1 6.67 7.547 0.8784 A 2 6.72 8.1991.4798 B 3 6.59 8.349 1.7588 B 4 6.56 7.79 1.2261 B 5 6.57 8.6 2.0339 B6 6.66 7.744 1.0811 C 7 6.67 7.928 1.2563 C 8 6.74 8.269 1.5299The target was heat killed MTB. The stock was at 245,000 cfu/ml anddiluted to 3000 cfu per ml of sputum. 12.3 ul per ml sputum. See belowfor amounts added.To the sputum samples was added 3 times the sputum volume as LB-EtOH-20%EDBE

tube sample ml ml LB ul Target total vol 1 0.8784 2.6352 10.80432 3.51362 1.4798 4.4394 18.20154 5.9192 3 1.7588 5.2764 21.63324 7.0352 4 1.22613.6783 15.08103 4.9044 5 2.0339 6.1017 25.01697 8.1356 6 1.0811 3.243313.29753 4.3244 7 1.2563 3.7698 15.45249 5.0252 8 1.5299 4.5897 18.817776.1196

Tubes were put in the heat block set at 80 C and incubated 70 minutes.The extractions were carried out using the Abbott PlexIDsp as describedabove. The reagents used are described above. After the samples haveincubated, add the lysates to the extraction plate. Maximum load was 5ml.

well load 1 3.5136 2 5 3 5 4 4.9044 5 5 6 4.3244 7 5.0252 8 5Nucleic acid (DNA) content was measured using the Nanodrop Lite AM03366in B130.

ml sputum vol recovered conc ng/ul ug DNA A260 260/280 0.88 200 569.3113.86 11.386 1.75 1.25 200 343.6 68.72 6.87 1.75 1.25 200 39.8 7.960.796 1.79 1.22 200 78.2 15.64 1.564 1.7 1.25 200 25.6 5.12 0.512 1.691.08 200 50.9 10.18 1.018 1.7 1.25 200 324.3 64.86 6.4886 1.87 1.25 200334.2 66.84 6.684 1.88

The extracted samples were tested using a PCR test for MTB DNA. FIG. 10Ashows results combined from FIGS. 10B-E. FIG. 10B shows results of thenegative control and 30,000 copies (positive control). FIG. 10C showsresults for Sputum sample A, 4 replicate assays of each of the twoextractions. The high positive control sample is also shown. FIG. 10Dshow results for Sputum sample B, 4 replicate assays of each of the fourextractions. The high positive sample is also shown. FIG. 10E showsresults for Sputum sample C. 4 replicate assays of each of the twoextractions. The high positive sample is also shown. The LB-EtOH-EDBEmix can solubilize the sputum and extract the MTB DNA in one step.

Example 6

The identification of amine monomers suitable for extraction of nucleicacid from cellular materials. Several additional amine monomers wereidentified as being suitable for the extraction of nucleic acids (e.g.,DNA, RNA, etc.) from cellular source material including, but not limitedto fresh, fixed and FFPE materials. Amine monomers tested were chosenbecause they appeared to be less hazardous that the EDBE solvent andhave similar properties to EDBE. The initial screening was done todetermine if any solvents would work in the extraction of yeast (C.alibicans) and S. aureus DNA from whole blood. CASA (C. albicans and S.aureus sample mixture; see, below) was used as a control. EDBE was ableto extract DNA from FFPE material and from the listed targets in wholeblood. This example tested the 7 amine monomers listed below andcompared to extraction with EDBE.

Solvents 1,5-Diamino-2-methylpentane Sigma- 329665-25 ml 15520-10-2Aldrich 2-(2-Aminoethoxy)ethanol Sigma- A54059-100 g 929-06-6 Aldrich2,2′-Ethylene- Sigma- 385506-500 ml 929-59-9 dioxy)bis(ethylamine)Aldrich 2-Amino-1-butanol Sigma- A43804-100 ml 96-20-8 Aldrich2-amino-2-methyl-1-propanol Sigma- A9199-100 ml 124-68-5 Aldrich2-amino-6-methylheptane Sigma- D161292-25 g 543-82-8 Aldrich3-Amino-1-propanol Sigma- A76400-100 g 156-87-6 Aldrich Amino-2-propanolSigma- 110248-100 ml 78-96-6 Aldrich

Lysis buffer (LB: see, above) and ethanol were mixed at a 2:1 ratiomaking the mix 33.3% ethanol (EtOH). Wash 1 was 50% EtOH. Wash 2 wasabout 74% EtOH. Samples were diluted to 200 cfu/ml of C. albicans or S.aureus. Blood was added to target samples 9:1. CASA standard was addedat 18 μl (CASA standard contains 100,000 cfu·ml C. albicans and 100,000cfu·ml S. aureus) in negative diluent (formulation designed to mimic thecomposition of plasma; Abbott Molecular product code #60217; AbbottPark, Ill.). Three replicants of each were tested along with an LB-EtOHcontrol and an NaOH control.

Extractions

3 replicants of each mix. 1 LB-EtOH LB-EtOH 2 NaOH NaOH 32,2′-Ethylenedioxy)bis(ethylamine) EDBE 4 2-Amino-1-butanol AB 52-(2-Aminoethoxy)ethanol AEE 6 2-amino-6-methylheptane AMH 72-amino-2-methyl-1-propanol AMP 8 3-Amino-1-propanol 3A1P 9Amino-2-propanol A2P 10 1,5-Diamino-2-methylpentane DMP #1 1.5 mlLB-EtOH #2 1.5 ml LB-EtOH + 30 ul 5M NaOH #3 1.3 ml LB-EtOH + 200 ulEDBE #4 1.3 ml LB-EtOH + 200 ul AB #5 1.3 ml LB-EtOH + 200 ul AEE #6 1.3ml LB-EtOH + 200 ul AMH #7 1.3 ml LB-EtOH + 200 ul AMP #8 1.3 mlLB-EtOH + 200 ul 3A1P #9 1.3 ml LB-EtOH + 200 ul A2P #10 1.3 mlLB-EtOH + 200 ul DMP

First run. Fifteen 2 ml tubes with 1.5 ml of the above reagents (3replicates of each mix). Fresh test samples were made and 50 μl ofsample was added to each tube. Samples were incubated at 58° C. for 4hours. Cassettes were set up for PCR in the Maxwell (Promega, MadisonWis.) with 50 μl MMP in each well. After lysis, the samples weredecanted into the lysis well and processed as follows: the samples werewashed 2× in Wash 1 and 2× in Wash 2. The washed MMP were eluted with100 μl elution solution.

Second run. The samples were lysed at 80° C. for 45 minutes. Thereminder was the same as the first run.

After lysis and processing PCR was performed on each sample as follows.

μl per C. albicans assay 25 μl Rx 50 Rx Primer IDT #42562400 - CandidaNC-009782 0.075 3.75 Primer for: 5′- TGCGATACGTAATATGAATTGCAGAT [SEQ IDNO: 1] Primer IDT #42562401 - Candida NC-009782 0.075 3.75 Primer rev:5′-CCAGAGGGCGCAATGTG [SEQ ID NO: 2] Probe 185896025-1 Candida TaqMan MGBProbe: 0.05 2.5 FAM-TGAATCATCGAATCTTTGAAC-MGB [SEQ ID NO: 3] 2X TaqmanBuffer AB #42562400 12.5 625 10X IPC mix AB #4308332 2.5 125 50X IPCtemplate AB#4304662 0.5 25 Total vol 15.7 sample 9

μl per S. aureus assay 25 μl Rx 50 Rx Primer IDT #39048657 - S. aureusNC-009782 0.075 3.75 Primer for: 5′- CATGGTTGACGATGAAGAATTATTAGA [SEQ IDNO: 4] Primer IDT #39233248 - S. aureus NC-009782 0.057 3.75 Primer rev:5′- TGGGAAGTCATATTCGCTTAATAAGTC [SEQ ID NO: 5] Probe 18583685-1 - S.aureus TaqMan MGB Probe: 0.05 2.5 5′-FAMAGTAGAAATGGAAGTTCG-MGB [SEQ IDNO: 6] 2X TaqMan Buffer AB#42562400 12.5 625 10X IPC mix AB#4308332 2.5125 50X IPC template AB #4304662 0.5 25 Total vol 15.7 Sample 9

FIG. 22 shows results of a one-way analysis of the data by solvent forC. albicans and S. aureus, respectively. FIG. 22 shows the FAM CT valuesgenerated from C. albicans and S. aureus cells extracted from wholeblood. Blood samples were mixed with a stock of both C. albicans and S.aureus (CASA samples) and extracted in the same reaction. C. albicans isa pathogenic yeast and S. aureus is a pathogenic gram positive bacteria.Although the cell walls of the two organisms differ in structure andcontent, they are both know to be difficult to lyse for DNA extraction.FIG. 22 A shows the results for the extraction of C. albicans usingvarious solvents added to the Abbott lysis buffer. The C. albicanssignal is improved (a lower CT value) under all conditions that have thevarious amine solvents added when compared to the results seen usingLB-EtOH. LB-EtOH is the Abbott lysis buffer from the m2000SamplePreparation SystemDNA extraction kit that has 33% ethanol added. Theaddition of NaOH to the extraction does appear to improve the extractionsomewhat but not to the extent seen with the amine solvents. The secondgraph shows the results obtained with S. aureus extracted from wholeblood under the same conditions as above. Again, in this case all theamine solvents improved the extraction but the use of NaOH also improvedthe extraction more than was seen with C. albicans. The graphs have astatistical analysis of the data on the right hand side. Circles that donot touch are considered to be statistically different from each other.As can be seen in both graphs the LB-EtOH extraction that has no addedcomponents is the least effective method (having the highest Ct value)and is statistically different than the other methods. Additional datais provided below.

Means and Std Deviations C. albicans

Std Err Lower Upper Level Number Mean Std Dev Mean 95% 95% 3A1P 332.4267 0.041633 0.02404 32.323 32.530 A2P 3 32.6667 0.406981 0.2349731.656 33.678 AB 3 33.7333 0.322542 0.18622 32.932 34.535 AEE 3 33.35670.092376 0.05333 33.127 33.586 AMH 3 32.9667 0.137961 0.07965 32.62433.309 AMP 3 33.6900 0.409512 0.23643 32.673 34.707 DMP 3 32.27000.115326 0.06658 31.984 32.556 EDBE 3 32.8733 0.298385 0.17227 32.13233.615 LB-EtOH 3 38.5300 0.596574 0.34443 37.048 40.012 NaOH 3 36.25670.352751 0.20366 35.380 37.133Means and Std Deviations S. aureus

Std Err Lower Upper Level Number Mean Std Dev Mean 95% 95% 3A1P 335.5733 0.306649 0.17704 34.812 36.335 A2P 3 34.7933 0.355012 0.2049733.911 35.675 AB 3 34.7267 0.100167 0.05783 34.478 34.975 AEE 3 34.93670.656531 0.37905 33.306 36.568 AMH 3 35.5500 0.838272 0.48398 33.46837.632 AMP 3 34.0967 0.351046 0.20268 33.225 34.969 DMP 3 33.90330.221435 0.12785 33.353 34.453 EDBE 3 34.6433 0.106927 0.06173 34.37834.909 LB-EtOH 3 36.7567 0.479618 0.27691 35.565 37.948 NaOH 3 34.47000.294449 0.17000 33.739 35.201

3A1P, A2P and DMP worked best for C. alibicans showing about a five CTimprovement over the LB-EtOH control. All were slightly better thanEDBE. AMP, DMP and NaOH worked well with S. aureus. Further studiesconfirmed efficacy of DMP, AEE, 2A1B, 3A1P and A2P for extraction of DNAfrom the recited test samples (data not shown) showing broadapplicability to the present invention with regard to amine monomersbeing effective for DNA extraction. Further still, a broad range ofconditions relating to temperature and time were found suitable for usewith the present invention although some temperatures and times gavebetter results (data not shown).

Example 7

3-amino-1-propanol (3A1P) extraction of FFPE samples. Three types ofsamples were tested. CRC (colorectal cancer), melanoma and lung tissues.Samples were compared to FFPE extraction systems known in the art andavailable from Qiagen (Valencia, Calif.) and Promega (Madison, Wis.).Sample sections (5 micron thick, not mounted on glass slides) from theparaffin block were taken sequentially such that every third sampletested the same extraction condition in order to lessen and sectionvariability. Lysis conditions for this experiment for the method of thepresent invention (3A1P extraction) are as follows. Lysis conditions:60% lysis buffer (LB); 20% EtOH and 20% 3A1P, total volume 1.5 ml.Incubations were tested at 78° C. and 94° C. and for time periods of 30minutes to 4 hours. After lysis incubation MMP (25 μl) were added tosamples. Capture time was 10 minutes at room temperature (RT). MMP werewashed 1× in 0.5 ml LB and 33% EtOH for 2 minutes at RT followed by 2×70% EtOH for 2 minutes each at RT. Samples were dried for 5 minutes andeluted with 100 μl of DI water for 10 minutes at 70° C. Samplesprocessed with the Qiagen and Promega protocols were processed accordingto manufacturer's directions. PCR was used for detection of BRAF-E. TheBRAF-E assay detects a mutation in the gene with FAM and the normal genewith CYC5. Ct values, a relative measure of concentration of target inthe PCR reaction, as is known to one of ordinary skill in the art, weredetermined. Ct data is shown in FIG. 11 for CRC samples and delta-Ct(dCt: change in Ct values between controls and test samples) are shownin FIG. 12. A value of less than 13 for dCt is considered to be positivefor the tested tumor marker. As can be seen from the data, the amineextraction procedure of the present invention is at least as good if notbetter than the art Qiagen and Promega methods while requiring fewerhandling steps and with faster processing time. In greater detail, inFIG. 11 the cycle threshold values (CT values) for the two signals areillustrated. The cycle threshold value refers to the cycle number in thePCR reaction where the signal is significantly above background. Thegreater amount of target in the assay allows the signal to be generatedwith a lower number of cycles so lower numbers indicate a higher amountof target. This figure shows the results from two separate blocks ofFFPE material from colorectal cancer tissue, C9 and C13. Serial sectionswere made from the blocks and separate sections represented by thenumbers were extracted with three different methods. The FAM signals areblue columns and the CY5 signals are red columns. The signals from theQiagen processed sections are represented by light blue and red columnsand labeled Qia. The signals from the Promega processed sections arerepresented by dark blue and red columns and labeled CSC. Both of thesemethods use protease digestion in the isolation of the DNA from the FFPEtissue. The signals from the Abbott amine solvent system are representedby bright blue and red columns. As can be seen in the graph, the signalsfrom the Abbott extractions are comparable to those obtained with theother two methods. FIG. 12 shows the difference between the FAM and CY5signals in FIG. 11. This dCT value is used to help determine if thetissue extracted is cancerous or not. A difference of less than 13indicates that there is a relatively higher amount of the mutant gene inthe sample (a lower CT value) and the sample may be cancerous. The threedifferent methods are illustrated by light green, dark green, and brightgreen columns for the Qiagen, Promega, and Abbott methods respectively.In both the C9 and C13 samples the Abbott methods gives a dCT valuecomparable to the other two methods.

In FIG. 13 and FIG. 14 the cycle threshold values (CT values) and thedCT values are shown for Lung tumor FFPE tissue. This figure shows theresults from two separate blocks of FFPE material from lung tumortissue, L1 and L4. Again, serial sections were made from the blocks andseparate sections represented by the numbers were extracted with threedifferent methods. The FAM signals are blue columns and the CY5 signalsare red columns. The signals from the Qiagen processed sections arerepresented by light blue and red columns and labeled Qia. The signalsfrom the Promega processed sections are represented by dark blue and redcolumns and labeled CSC. Both of these methods use protease digestion inthe isolation of the DNA from the FFPE tissue. The signals from theAbbott amine solvent system are represented by bright blue and redcolumns. As can be seen in the graph, the signals from the Abbottextractions are comparable to those obtained with the Promega system andone of the samples (L1) processed with the Qiagen method. However the L4sample processed the Qiagen method did not isolate DNA as well as thePromega or the Abbott method. FIG. 14 shows the difference between theFAM and CY5 signals in FIG. 13. The three different methods areillustrated by light green, dark green, and bright green columns for theQiagen, Promega, and Abbott methods respectively. In both the L1 samplesthe Abbott methods gives a dCT value comparable to the other two methodwhole the L4 sample did not appear to be extracted as well with theQiagen method.

The extraction protocol experiment was duplicated on melanoma samples.Extraction and processing conditions were as above. FIG. 15 shows Ctdata and FIG. 16 shows dCt data. In FIG. 15 the cycle threshold values(CT values) for the two signals are illustrated. The cycle thresholdvalue refers to the cycle number in the PCR reaction where the signal issignificantly above background. The greater amount of target in theassay allows the signal to be generated with a lower number of cycles solower numbers indicate a higher amount of target. This figure shows theresults from two separate blocks of FFPE material from melanoma tissue,M11 and M14. Serial sections were made from the blocks and separatesections represented by the numbers were extracted with three differentmethods. The FAM signals are blue columns and the CY5 signals are redcolumns. The signals from the Qiagen processed sections are representedby light blue and red columns and labeled Qia. The signals from thePromega processed sections are represented by dark blue and red columnsand labeled CSC. Both of these methods use protease digestion in theisolation of the DNA from the FFPE tissue. The signals from the Abbottamine solvent system are represented by bright blue and red columns. Ascan be seen in the graph, the signals from the Abbott extractions arecomparable to those obtained with the other two methods. FIG. 16 showsthe difference between the FAM and CY5 signals in FIG. 15. This dCTvalue is used to help determine if the tissue extracted is cancerous ornot. A difference of less than 13 indicates that there is a relativelyhigher amount of the mutant gene in the sample (a lower CT value) andthe sample may be cancerous. The three different methods are illustratedby light green, dark green, and bright green columns for the Qiagen,Promega, and Abbott methods respectively. In the M11 samples the Abbottmethods appears to give a better dCT value (lower dCT) than the othertwo methods. The M14 samples show an interesting pattern in that somesections of the tissue have a high dCT value but as the sections gofurther into the sample, they lower. This indicates that some sectionsof a tissue block may not contain tumor cells and is necessary to testmultiple sections. The ability to isolate DNA from multiple sections isdiscussed below.

These three experiments show that the amine solvent extraction processof the present invention performs as least as well as the Qiagen andPromega protocols while requiring fewer handling steps and with fasterprocessing time.

Example 8

The versatility of the compositions and methods of the present inventionprovide for improvements over prior art procedures. For example, forslide mounted specimens, the prior art procedures require the scrapingof sample off of the glass slide. This step is subject to operatorerror. The required scraping is time consuming, used sharp instrumentsand has a high probability of cross contamination. The compositions andmethods of the present invention allow for the extraction of the DNAdirectly from the slide without scraping the sample from the slide.Further, multiple slides can be processed together to alleviate possibleassay variation caused by section variation (i.e., “hit or miss” causedby variation between sample sections) or to help detect low leveltargets. Slides can be processed in reception vessels (RV) designed forholding multiple slides. The DNA in the sample has a greater affinityfor the MMP than for the glass slide (this may be the result ofdifferent types of glass and/or MMP added to excess). FIG. 17 shows agraph with results of processing with the compositions and methods ofthe present invention for a hepatitis B virus control and FIG. 18 showsthe same procedure with MMP added during the lysis-incubation procedure.Addition of the MMP at this point in the protocol results in the captureof the DNA on the MMP. An experiment conducted on breast FFPE slidesproves the effectiveness of this procedure. Samples were extracted witheither LB-EtOH-3A1P or LB-3A1P with EtOH added after incubation. Thesamples were also incubated with or without MMP. The incubation was at90° C. for 2 hours and 20 minutes for each condition. FIG. 19 shows thatthe addition of MMP in the lysis incubation, with or without EtOH,resulted in binding of the DNA to the MMP as shown by the dRn values.dRn stands for delta normalized response which is the value for thefluorescent signal from the PCR reaction after it has been baselinedusing a program called Multianalyze4 (Abbott Molecular in-house softwareprogram, Abbott Park, Ill. Other suitable programs are commerciallyavailable, as is known to one of ordinary skill in the art as isevidence by teachings at web sites such aswww.gene-quantification.de/hkg.html, and similar). The CT value isgenerated from the point where the dRn crosses a particular thresholdvalue for the dRn.

Example 9

In this example multiple slide were processed simultaneously. 1 to 4slides were processed in the same reaction vessel. Blank slides wereused as placeholders in conditions where fewer than 4 slides wereprocessed. The processing of more than one slide at a time may help inthe detection of low copy number targets. FIG. 20 shows the results ofprocessing from 1 to 4 slides simultaneously. Detection was improvedwith each additional slide processed. Both breast tissue FFPE andPathVysion-A probe check normal slides (Abbott Laboratories, AbbottPark, Ill.) were tested. FIG. 21 shows a one-way ANOVA analysis of thedata.

In greater detail, FIG. 20 is an illustration of the amplificationcurves generated when 1 to 4 slides containing FFPE material wereextracted in the same reaction vessel. Two different sets of slides wereused. One set consisted of breast tissue FFPE slides that stillcontained the paraffin. The other set contained PathVyson-A Probe ChekNormal slides (FFPE treated cells) that had been tested using the FISHprocess and had the paraffin removed during the FISH processing. FIG. 21has two graphs. The first shows the CY5 CT values for the both sets ofextractions. B1, B2, B3, and B4 contained 1, 2, 3, and 4 breast tissueslides respectively. The CT value decreases with each additional slidewhich indicates that more DNA was in the amplification reaction with agreater number of slides. The level does not decrease after 3 slides andmay indicate that a maximal level of material was extracted at thatpoint. The MR value stands for MaxRatio and indicates the amplitude ofthe amplification reaction. A higher MR value indicates that theamplification reaction is more robust. The MR value does decrease withthe breast tissue samples with 4 slides and this may indicate that themaximal level of material was extracted with 3 slides of this tissue orthat the increased level of paraffin may be an issue. The material fromthe FISH processed slides, PV1 to PV4, also shows a decrease in the CTvalue with a greater number of slides and thus more DNA in the reaction.The CT values continue to decrease with the fourth slide. The MR valuesdo not decrease with the added slides. This set of slides does not haveparaffin and that may be reflected in this data.

Example 10

In this example, slides previously processed for fluorescent in situhybridization (FISH) analysis are processed with the compositions andmethods of the present invention. The results show that nucleic acid isextracted from the slides previously processed for FISH analysis andthat the isolated DNA is suitable for further analysis after extractionfrom the FISH process slides.

Example 11

Extraction of nucleic acid from Mycobacterium tuberculosis (MTB). Lysiswith the 3A1P lysis composition will dissolve sputum and extract nucleicacid from MTB. Further, this composition may be used to inactivate thetarget MTB. While not wishing the present invention to be limited bytheory, it is believed that the high pH of the 3A1P lysis composition ofthe present invention may be responsible for the inactivation of thetarget MTB.

I claim:
 1. A method of isolating an extraction solution comprisingnucleic acids from yeast in a whole blood sample, said methodcomprising: a) providing i) a whole blood sample suspected of comprisingyeast and ii) an aqueous extraction solution comprising an aminemonomer, a chaotrope, and a detergent, wherein said amine monomer isselected from the group consisting of is2,2′-(ethylenedioxy)bis(ethylamine) (EDBE), 1,3-diaminopropane and3-amino-1-propanol and wherein the concentration of amine monomer insaid aqueous extraction solution is about 30% to about 50% and whereinthe concentration of said detergent is about 8% to about 15%; b)contacting said whole blood sample suspected of comprising yeast withsaid extraction solution resulting in lysis of the yeast and theisolation of an extraction solution comprising nucleic acids.
 2. Themethod of claim 1, wherein said amine monomer is a primary aminemonomer.
 3. The method of claim 2, wherein said amine monomer is2,2′-(ethylenedioxy)bis(ethylamine) (EDBE).
 4. The method of claim 2,wherein said amine monomer is 1,3-diaminopropane.
 5. The method of claim2, wherein said amine monomer is 3-amino-1-propanol.
 6. The method ofclaim 1, wherein said chaotrope is selected from at least one of thegroup consisting of urea, and guanidine thiocyanate (GITC).
 7. Themethod of claim 6, wherein the concentration of the chaotrope in saidaqueous extraction solution is about 4 M to about 5 M.
 8. The method ofclaim 1, wherein said detergent is selected from at least one of thegroup consisting of a polysorbate, deoxycholate, sodium deoxycholate,sodium dodecyl sulfate (SDS), nonyl phenoxypolyethoxylethanol andpolyethylene glycol.
 9. The method of claim 1, further comprising analcohol wherein said alcohol is selected from at least one of the groupconsisting of ethanol and butanol.
 10. The method of claim 9, whereinthe concentration of said alcohol is about 15% to about 25%.
 11. Themethod of claim 1, wherein said sample is selected from living cellularsource material and fixed cellular source material.
 12. The method ofclaim 11, wherein said living cellular source material comprises asuspension of single cells.
 13. The method of claim 11, wherein saidsample comprises formalin-fixed paraffin embedded (FFPE) material. 14.The method of claim 1, further comprising wherein said aqueousextraction solution is enzyme-free.
 15. The method of claim 1, furthercomprising wherein said aqueous extraction solution is protease-free.16. The method of claim 1, further comprising wherein said nucleic acidsare isolated from an extraction solution comprising nucleic acids withadditional steps, the additional steps comprising i) adsorbing saidnucleic acids onto silica coated magnetic particles by introducing saidparticles into said extraction solution containing extracted nucleicacids to create nucleic acid adsorbed silica coated magnetic particles;ii) washing said nucleic acid adsorbed silica coated magnetic particles,iii) eluting said nucleic acids from said silica coated magneticparticles into the elution solution and; iv) isolating said nucleicacids from said elution solution.
 17. The method of claim 1, whereinsaid method excludes mechanical disruption of the yeast.